A recombinant humanized collagenase type Ⅲ cleavage peptide-sodium alginate oxidation complex and a preparation method and application thereof

Abstract

The application discloses a recombinant type III humanized collagenase cut peptide-sodium alginate oxidation complex and a preparation method and application thereof. The complex is formed by cross-linking and compounding of recombinant type III humanized collagen treated by specific protease and oxidized sodium alginate. The specific protease is selected from one or more of Lys-C, Glu-C, protease K, pepsin and trypsin. The recombinant type III humanized collagen is treated by enzyme cutting to form specific polypeptide fragments which can be cross-linked with the oxidized sodium alginate to form the complex with the modified sodium alginate polysaccharide molecular chain as the main chain and the recombinant type III humanized collagenase cut polypeptide as the auxiliary chain, so that the adhesion of the recombinant type III humanized collagen in the skin is improved, the action time of the recombinant type III humanized collagen on the skin surface is prolonged, the utilization of the cell layer is more favorable, and the biological repair ability and moisturizing performance of the recombinant type III humanized collagen are improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a recombinant type III humanized collagenase-oxidized sodium alginate complex, its preparation method, and its application. Background Technology

[0002] Collagen is the most abundant protein in the human body, accounting for more than 30% of total protein. Type III collagen, a key fibroblast collagen, is widely distributed in connective tissues such as skin, lungs, liver, intestines, and blood vessels, participating in the regulation of biological processes such as cell adhesion, proliferation, and migration, and playing a crucial role in the repair of damaged skin. In recent years, advancements in recombinant gene technology have enabled the high-purity and high-specificity production of human type III collagen without the use of animal sources, laying the foundation for its application in medical devices and skincare products. Recombinant humanized type III collagen (rhColⅢ) is a novel biomaterial synthesized through genetic engineering, highly consistent with the structure and function of natural human type III collagen. Because it is prepared using a human gene expression system and does not contain non-human amino acid sequences, it avoids animal-derived contamination and immune rejection reactions, showing great potential in wound repair, tissue regeneration, medical aesthetics, and drug delivery. Recombinant type III humanized collagen microspheres (hereinafter referred to as rhColIII microspheres) are microprotein spheres with a cavity structure formed by cyclizing linear rhColIII folds using cyclase. Because they retain the triple helix structure and bioactivity consistent with human natural collagen, and effectively slow down the degradation rate of proteases, they improve stability and durability in human tissues, and have good application prospects in medical materials, daily chemical cosmetics and food.

[0003] The research on recombinant type III humanized collagen in skin repair and anti-aging has long been a focus of the medical and skincare industries. Studies have shown that rhCol III can promote fibroblast proliferation, migration, and collagen deposition, accelerating the healing of acute and chronic wounds (such as diabetic ulcers, burns, and oral ulcers). Simultaneously, it can also improve skin elasticity, reduce wrinkles, and improve UV-induced skin damage by regulating collagen metabolism and antioxidant mechanisms, demonstrating excellent application prospects. Currently, rhCol III has been approved as a Class III medical device for facial filling and improving dynamic wrinkles. Products include freeze-dried fibers and injectable solutions, widely used in dermatology, gynecology, proctology, and other fields. However, both rhColIII and rhColIII microspheres are single-component collagen, making it difficult to meet the demands of multiple bioactivities. For example, in applications involving skin and mucous membrane damage repair, they struggle to achieve lasting effects in skin repair and moisturizing. Furthermore, due to the high cost of rhColIII raw materials, the concentration used in general products is often low. This makes it difficult for recombinant collagen molecules to form a continuous fibrous network or gel structure at the application interface (such as skin wounds), thus affecting their adhesion to cells. In particular, after cyclization, the particle size of rhColIII microspheres is only 7-8 nanometers, further reducing their skin adhesion and severely impacting their biorepair capabilities.

[0004] Currently, the main method for improving the skin adhesion of collagen is cross-linking. For example, rhColⅢ is chemically modified and then cross-linked stepwise with carboxymethyl chitosan to form collagen derivatives. These derivatives are then mixed with moisturizing ingredients to create composite biological dressings with good repair and moisturizing effects. Alternatively, stacked short sequences are repeatedly combined with continuous short sequences of human collagen amino acids with a triple helix structure to form specific recombinant collagen, which is then cross-linked with carboxymethyl chitosan and sodium hyaluronate to improve the biological properties of the prepared medical dressings. However, modifying recombinant collagen with chemical reagents and adding cross-linking catalysts carries a high risk of introducing impurities, and the process is cumbersome and lacks scalability benefits.

[0005] Sodium alginate (SA) is a naturally occurring anionic polysaccharide found in the cell walls of brown algae. It is composed of two units, β-D-mannuronic acid (M) and α-L-guluronic acid (G), randomly arranged or linked in segments by 1,4-glycosidic bonds. It is readily soluble in water, forming a highly viscous, pseudoplastic solution. When encountering polyvalent cations such as calcium, strontium, and barium, the G segment forms an "eggbox" coordination, instantly cross-linking into an irreversible hydrogel. This mild, room-temperature, solvent-free gelation characteristic makes it a classic "ionic gel" material. Sodium alginate has good biocompatibility; its degradation products are oligosaccharides and alginic acid, which can be cleared by the body. It has low immunogenicity and is listed as a GRAS food additive by the US FDA, and is also approved for use in wound dressings and pharmaceutical preparations. There are also reports indicating that combining recombinant collagen with sodium alginate and other ingredients, and adjusting the pH to gel the recombinant protein, can yield dressing products with moisturizing and repairing effects. However, practice has shown that simple physical mixing cannot improve the repair effect of collagen, and moisturizing is only the contribution of sodium alginate hydrogel, which cannot achieve the purpose of improving the adhesion of collagen in the skin.

[0006] Therefore, how to enhance the adhesion of recombinant type III humanized collagen in the skin and improve its biological repair and moisturizing properties remains an urgent problem to be solved. Summary of the Invention

[0007] To address the skin adhesion issues associated with recombinant type III humanized collagen and to enhance the biorepair and moisturizing properties of recombinant type III humanized collagen microspheres, this invention provides a recombinant type III humanized collagen protease-coated peptide-oxidized sodium alginate complex, its preparation method, and its application. Recombinant type III humanized collagen and sodium alginate are mixed in a specific ratio and then subjected to enzymatic cleavage and cross-linking treatments. The resulting complex has a larger molecular weight and higher viscosity, significantly improving the adhesion rate of recombinant type III humanized collagen microspheres to skin wounds, thus promoting skin repair. The specific technical solution is as follows: First, this invention provides a recombinant type III humanized collagen protease-cleaved peptide-oxidized sodium alginate complex, which is a cross-linked compound formed by recombinant type III humanized collagen treated with specific protease cleavage and oxidized sodium alginate; wherein the specific protease used for the protease cleavage of the recombinant type III humanized collagen is selected from one or more of Lys-C, Glu-C, proteinase K, pepsin or trypsin. Glu-C is preferred.

[0008] The aforementioned recombinant type III humanized collagen protein cleavage peptide-oxidized sodium alginate complex, wherein the recombinant type III humanized collagen protein is recombinant type III humanized collagen protein microspheres, and its mass ratio with oxidized modified sodium alginate is 1:(10-20), preferably 1:20.

[0009] The aforementioned recombinant type III humanized collagenase cleavage peptide-oxidized sodium alginate complex has an oxidized sodium alginate with a molecular weight of 150kDa to 200kDa and a mass concentration of oxidized sodium alginate in the complex of 10g / L.

[0010] The aforementioned recombinant type III humanized collagenase cleavage peptide-oxidized sodium alginate complex has a specific protease activity of 100 IU / L to 200 IU / L, preferably 200 IU / L.

[0011] Secondly, this invention provides a method for preparing the aforementioned recombinant type III humanized collagenase-cleaved peptide-oxidized sodium alginate complex, comprising the following steps: Step 1: Dissolve recombinant type III humanized collagen and oxidized modified sodium alginate in phosphate buffer solution according to the following mass ratios; Step 2: Add a specific protease to carry out the enzymatic digestion reaction; Step 3: Adjust the pH of the reaction solution to 9-10 and the temperature to 55-65℃ to carry out the cross-linking reaction and obtain the enzymatically digested protein peptide-sodium alginate complex solution; Step 4: The enzymatically digested protein peptide-sodium alginate complex solution is subjected to ultrafiltration to remove impurities and concentration, while the solution is replaced until the pH value of the concentrate is within the specified range, thus obtaining the enzymatically digested protein peptide-sodium alginate complex concentrate. Step 5: Add water to the concentrated solution of the enzymatically digested protein peptide-sodium alginate complex to a certain volume.

[0012] As a preferred technical solution, in the aforementioned method for preparing the recombinant type III humanized collagenase cleavage peptide-oxidized sodium alginate complex, in step one, the concentration of the phosphate buffer solution is 0.01M; the preparation method of the oxidized modified sodium alginate is as follows: sodium alginate is dispersed in water, sodium periodate is added, and the mixture is reacted at 3-5°C in the dark for 15-18 hours, followed by the addition of ethylene glycol and the reaction at room temperature for 50-70 minutes to obtain a precipitate; then the precipitate is washed sequentially with water and anhydrous ethanol, and vacuum dried at room temperature for 6 hours; the mass ratio of sodium alginate, sodium periodate, and ethylene glycol is 1:0.1:5.

[0013] As a preferred technical solution, in the aforementioned method for preparing the recombinant type III humanized collagenase-oxidized sodium alginate complex, in step two, the enzyme digestion reaction time is controlled within 4 hours, the enzyme digestion reaction temperature is 37°C, and the reaction pH value is the optimal pH for the specific protease.

[0014] As a preferred technical solution, in the aforementioned method for preparing the recombinant type III humanized collagenase-oxidized sodium alginate complex, in step three, the solution for adjusting the pH of the reaction solution is a 6M NaOH solution; and the cross-linking reaction time is 1.5–3 hours.

[0015] As a preferred technical solution, in the aforementioned method for preparing the recombinant type III humanized collagenase cleavage peptide-oxidized sodium alginate complex, in step four, the ultrafiltration and concentration are performed by using a 10kDa ultrafiltration membrane to retain the filtrate and collect the filtrate; the solution replacement is performed by adding a 0.01M phosphate buffer solution with a pH of 6.8 during ultrafiltration to replace the solution system of the filtrate, and adjusting the pH of the retained concentrate to 7.0-8.0.

[0016] In addition, the present invention also provides an application of the aforementioned recombinant type III humanized collagenase cleavage peptide-oxidized sodium alginate complex, specifically for the preparation of products for adjuvant treatment of skin and mucous membrane damage repair, or as an effective ingredient in the preparation of adjuvant treatment products suitable for skin and mucous membrane damage repair.

[0017] The beneficial effects of this invention are: 1) This invention involves treating recombinant type III humanized collagen with specific protease to form specific polypeptide fragments, which are then cross-linked with oxidized sodium alginate to form a complex with modified sodium alginate polysaccharide molecules as the main chain and protease-cleaved polypeptides from recombinant type III humanized collagen as the auxiliary chain. This enhances the adhesion of recombinant type III humanized collagen in the skin. Furthermore, the enzymatically cleaved product is rich in polypeptides and biodegradable, making it more beneficial for cellular utilization and repair. Simultaneously, the enzymatically cleaved small-molecule collagen peptides can form a dense and uniform cross-linked network with oxidized sodium alginate (SA), enabling precise control of the degradation rate of recombinant type III humanized collagen, prolonging its action time on the skin surface, and further enhancing its bio-repair capabilities and moisturizing properties.

[0018] 2) The specific protease used in this invention is selected from one or more of Lys-C, Glu-C, Arg-C, and Trypsin. Choosing these enzymes for enzymatic digestion effectively controls the reaction conditions, ensuring specific degradation of collagen and obtaining collagen peptides with excellent biological activity. This effectively solves the problem of low activity in recombinant collagen in existing technologies. The recombinant type III humanized collagen of this invention is preferably recombinant type III humanized collagen microspheres. This not only avoids the immune rejection and allergy problems that may be caused by traditional animal-derived collagen, improving the safety and applicability of the product, but also ensures that polypeptide fragments with well-defined amino acid sequences are obtained after treatment with the selected specific enzymes, avoiding the introduction of impurities and ensuring the effectiveness of the enzymatically digested peptides combined with modified sodium alginate. Simultaneously, the molecular weight of the enzymatically digested peptides is clearly defined, facilitating product quality control. The molecular weight of oxidized sodium alginate is also preferably 150kDa to 200kDa, ensuring that the viscosity of the complex formed after cross-linking with the collagen peptides is suitable and that it achieves good skin adhesion.

[0019] 3) The preparation method of this invention involves oxidatively modifying sodium alginate, causing the ortho-dihydroxyl groups on its molecules to be oxidized into aldehyde groups. By adjusting the reaction temperature and pH value, the aldehyde groups of the oxidized sodium alginate are cross-linked with the amino terminus of the polypeptide fragments obtained from enzymatic digestion. The cross-linking reaction conditions are optimized to ensure the stability and uniformity of the composite, effectively improving the quality stability and controllability of the composite. Compared with simply mixing collagen with sodium alginate, this invention covalently cross-links the polypeptides formed by enzymatic digestion of recombinant type III humanized collagen with the aldehyde groups of oxidatively modified sodium alginate, resisting the degradation effect of collagenase, prolonging the retention time of the material in wounds or tissues, increasing its viscosity on the skin, and further enhancing its biorepair and moisturizing capabilities.

[0020] Overall, the composite of this invention has better moisturizing properties and cell activity, which can provide a good moist environment for skin wounds and promote rapid repair and healing of epidermal cells. Moreover, the preparation method of this invention uses ultrafiltration and concentration processes to purify the composite solution, effectively removing impurities and improving purity, while ensuring the biological activity of the composite, which has good practical value and application prospects. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the recombinant type III humanized collagenase cleavage peptide-oxidized sodium alginate complex of the present invention (wherein, the transverse main chain is sodium alginate, and the side chain is recombinant type III humanized collagenase cleavage peptide). Figure 2 The recombinant type III humanized collagenase cleavage peptide-oxidized sodium alginate complex prepared in Example 1 of this invention; Figure 3Example 1 shows the molecular weight detection results of each complex. Figure 4 Example 1 shows the cell adhesion rate test results for each complex. Figure 5 The results of cell migration tests for each complex are shown in Example 1 of the present invention. Detailed Implementation

[0022] To address the issues of recombinant type III humanized collagen's single component, its inability to meet the needs of multiple biological activities, its low adhesion rate to skin wounds, and its difficulty in achieving lasting effects in skin repair and moisturizing, this invention provides a recombinant type III humanized collagen protease-cleaved peptide-oxidized sodium alginate complex and its preparation method. The method involves specifically enzymatically cleaving recombinant type III humanized collagen and then cross-linking it with oxidically modified sodium alginate to prepare a complex with a larger molecular weight and higher viscosity. Figure 1 As shown, it significantly improved the adhesion rate of recombinant type III humanized collagen microspheres to skin wounds, which is beneficial to skin repair.

[0023] Specifically, in the recombinant type III humanized collagen protease-cleaved peptide-oxidized sodium alginate complex provided by this invention, the specific protease used for the protease cleavage treatment of the recombinant type III humanized collagen is selected from one or more of Lys-C, Glu-C, proteinase K, pepsin, or trypsin. These enzymes can cleave recombinant type III humanized collagen to different degrees, and the enzyme activity of the system is controlled at 100 IU / L to 200 IU / L. Among them, Lys-C, Glu-C, and Arg-C cleave lysine (K), glutamate (E), and arginine (R), respectively; while trypsin mainly cleaves lysine (K) and arginine (R). The recombinant type III humanized collagen is composed of recombinant type III humanized collagen microspheres. Based on its amino acid sequence characteristics, a combination of Lys-C and Arg-C is used to cleave the target protease into 10 peptide fragments, with an average of 7 amino acids per fragment. Glu-C and trypsin are used separately. Glu-C can cleave the target protease into 6 fragments, with an average of 12 amino acids per fragment, while trypsin can cleave the target protease into 10 fragments, with an average of 7 amino acids per fragment. The larger the amino acid content, the higher the viscosity of the complex formed by cross-linking with oxidized sodium alginate, and the better the skin adhesion effect. Therefore, Glu-C is the preferred specific protease. The molecular weight of the oxidized sodium alginate is 150kDa to 200kDa, and the mass concentration of oxidized sodium alginate in the complex is 10g / L. The mass ratio of the recombinant type III humanized collagen microspheres to oxidized sodium alginate is 1:(10-20), preferably 1:20.

[0024] The preparation method of the recombinant type III humanized collagenase cleavage peptide-oxidized sodium alginate complex of the present invention includes the following steps: Step 1: Dissolve recombinant type III humanized collagen and oxidized modified sodium alginate separately in phosphate buffer at a mass ratio of 0.01M. The oxidized modified sodium alginate is prepared by dispersing sodium alginate in water, adding sodium periodate, reacting at 3-5°C in the dark for 15-18 hours, then adding ethylene glycol and reacting at room temperature for 50-70 minutes to obtain a precipitate. The precipitate is then washed sequentially with water and anhydrous ethanol, and vacuum dried at room temperature for 6 hours. The mass ratio of sodium alginate, sodium periodate, and ethylene glycol is 1:0.1:5.

[0025] Step 2: Add a specific protease to carry out the enzymatic digestion reaction; the digestion reaction time is controlled within 4 hours, the digestion reaction temperature is 37℃, and the reaction pH value is the optimal pH of the specific protease.

[0026] Step 3: Adjust the pH of the reaction solution to 9-10 and the temperature to 55-65℃ to carry out the cross-linking reaction and obtain the enzymatically digested protein peptide-sodium alginate complex solution; the solution used to adjust the pH of the reaction solution is a 6M NaOH solution; the cross-linking reaction time is 1.5-3 hours.

[0027] Step 4: The enzymatically digested protein peptide-sodium alginate complex solution is subjected to ultrafiltration for impurity removal and concentration, while simultaneously undergoing solution replacement until the pH value of the concentrate reaches the specified range, thereby obtaining the enzymatically digested protein peptide-sodium alginate complex concentrate. The ultrafiltration for impurity removal and concentration is performed using a 10kDa ultrafiltration membrane for retention, and the retained liquid is collected. The solution replacement is performed by adding a 0.01M phosphate buffer solution with a pH of 6.8 during ultrafiltration to replace the retained liquid solution system, adjusting the pH value of the retained concentrate to 7.0-8.0.

[0028] Step 5: Add water to the concentrated solution of the enzymatically digested protein peptide-sodium alginate complex to a certain volume.

[0029] The recombinant type III humanized collagenase-cleaved peptide-oxidized sodium alginate complex provided by this invention can be used as an adjunct treatment for the repair of skin and mucous membrane damage, or as an effective ingredient in the preparation of adjunct treatment products suitable for the repair of skin and mucous membrane damage.

[0030] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with embodiments.

[0031] The 0.01M phosphate buffer used in the following examples was prepared as follows: Weigh 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4 respectively; dissolve all substances in 800ml distilled water; adjust the pH of the solution to 7.2 with NaOH; and finally add distilled water to a final volume of 1L.

[0032] Example 1 This embodiment aims to prepare a recombinant type III humanized collagenase-cleaved peptide-oxidized sodium alginate complex, as detailed below: Step 1: Disperse 1 part of sodium alginate with a molecular weight of 150kDa to 200kDa in water, add 0.1 part of sodium periodate, react at 4°C in the dark for 16 hours, then add 5 parts of ethylene glycol and react at room temperature for 1 hour. After the reaction is complete, a precipitate is obtained. Wash the precipitate with water and anhydrous ethanol in sequence, and then vacuum dry at room temperature for 6 hours to obtain oxidized sodium alginate. Dissolve recombinant type III humanized collagen microspheres and oxidized sodium alginate in 0.01M phosphate buffer at addition amounts of 0.5 g / L and 10 g / L, respectively.

[0033] Step 2: Add the specific protease Glu-C to make the enzyme activity of the system 200 IU, and carry out the enzymatic digestion reaction for 200 min. The temperature of the enzymatic digestion reaction is 37℃ and the pH of the reaction is 7.2. Step 3: Adjust the pH to 10 with 6M NaOH and adjust the temperature to 60℃, continue the reaction for 2 hours to carry out cross-linking; Step 4: Use a 10kDa ultrafiltration membrane to perform ultrafiltration to remove impurities, concentrate and retain the solution, and collect the retained liquid; while ultrafiltration, add 0.01M phosphate buffer (pH 6.8) to replace the solution until the pH reaches between 7.0 and 8.0; Step 5: Add water to the collected retentate to bring it to the original volume (the volume of the solution in Step 1), thus obtaining a recombinant type III humanized collagenase-oxidized sodium alginate complex solution with uniform molecular weight, denoted as Complex 1 of this invention. Figure 2 As shown.

[0034] Example 2 This embodiment also aims to prepare a recombinant type III humanized collagenase-coated peptide-oxidized sodium alginate complex. The only difference between this embodiment and Example 1 is the specific protease used in step two; all other ratios and process parameters are the same as in Example 1. In step two of this embodiment, the added specific proteases are Lys-C and Arg-C, with an enzyme activity of 100 IU / L for both. The prepared recombinant type III humanized collagenase-coated peptide-oxidized sodium alginate complex solution is designated as Complex 2 of this invention.

[0035] Example 3 This embodiment also prepares a recombinant type III humanized collagenase-cleaved peptide-oxidized sodium alginate complex. The difference from Example 1 is that in step two, the specific protease used is trypsin, the enzyme activity is 200 IU / L, the enzyme digestion temperature is 37℃, and the reaction pH is 7.2; in step three, 6M NaOH is used to adjust the pH to 9 for cross-linking reaction; the remaining ratios and process parameters are the same as in Example 1. The prepared recombinant type III humanized collagenase-cleaved peptide-oxidized sodium alginate complex solution is designated as Complex 3 of this invention.

[0036] Example 1 This embodiment examines the molecular weight, viscosity, moisturizing properties, and cell viability of the recombinant type III humanized collagenase-oxidized sodium alginate complex prepared in Examples 1-3.

[0037] This embodiment uses a complex of recombinant type III humanized collagen and sodium alginate as a comparison. Specifically, it is prepared based on Example 1. The preparation method of the recombinant type III humanized collagen and sodium alginate complex solution differs from Example 1 in that it does not involve enzymatic digestion; that is, its preparation process does not include step two. Specifically, recombinant type III humanized collagen microspheres and sodium alginate with a molecular weight of 150kDa-200kDa are dissolved in 0.01M phosphate buffer at addition amounts of 0.5g / L and 10g / L, respectively. Then, the pH is adjusted to 10 using 6M NaOH, and the temperature is adjusted to 60℃, continuing the reaction for 2 hours to achieve cross-linking. The complex solution is then subjected to ultrafiltration to remove impurities and concentration. A 10kDa ultrafiltration membrane is used for retention, and 0.01M phosphate buffer (pH 6.8) is added during ultrafiltration to replace the solution until the pH reaches between 7.0 and 8.0. The retained solution is collected and diluted with water to the original volume. The prepared complex solution is denoted as the control complex.

[0038] 1) Molecular weight determination: Size exclusion chromatography was used with an Ultimate BIO-SEC300 column (7.8 mm * 300 mm), a mobile phase of 0.1 mol / L sodium nitrate solution, a flow rate of 0.5 ml / min, a column oven temperature of 40℃, an injection volume of 20 μL, and a differential detector. Samples were filtered through a 0.22 μm filter and diluted 100-fold with the mobile phase before injection. The molecular weight chromatograms of each complex are shown below. Figure 3 As shown in the figure (1-3 correspond to composites 1-3 of the present invention, and 4 is the comparative composite).

[0039] Because size exclusion chromatography is used, the earlier the peak time, the larger the molecular weight. Figure 3The test results show that the solution of complex 1 prepared in Example 1 had the earliest peak time, while the solution of the control complex had the latest peak time. This means that the complex molecules formed by the cross-linking of the protein peptides of complexes 1-3 prepared in Examples 1-3 after specific protease digestion with sodium alginate are all larger than the undigested control complex. This indicates that the peptides formed after enzymatic digestion can effectively cross-link with oxidized sodium alginate to form complexes, ensuring effective retention on the skin due to the large molecular viscosity of sodium alginate. The complex solution prepared using Glu-C protease digestion has the largest molecular weight, thus providing better skin adhesion.

[0040] In addition, by Figure 3 The test results also show that there are no impurities (no impurity peaks) in any of the complex solutions. This indicates that the preparation method of the present invention, through ultrafiltration, can filter out small molecule impurities, ensuring the purity of the complex and facilitating the later application of the product.

[0041] 2) Viscosity test: 200 ml of the complex solution was placed in a 250 ml beaker and measured using a rotational viscometer with rotor No. 1 at 3 rpm. The results are shown in Table 1.

[0042] Table 1: Viscosity test results of various recombinant type III humanized collagen and sodium alginate complexes

[0043] As can be seen from the results in Table 1, the viscosity test results are consistent with the molecular weight test results. The larger the molecular weight, the greater the viscosity and the better the adhesion performance in the skin.

[0044] 3) Moisturizing performance test, the method is as follows: ①30 test subjects were tested. The specific test area (3cm*3cm) on the inside of the human forearm was kept clean and the test subjects sat quietly indoors for 30 minutes. ②The baseline skin moisture level of the subjects was measured using the Courage+Khazaka skin moisture meter; ③ The subject applies the test sample to the test area and starts timing 3 seconds later. The samples from top to bottom are the composites of the present invention prepared in Examples 1-3 and the comparative composite prepared in Effect Example 1. The timing is 15 minutes.

[0045] ④ After the timer expires, gently wipe the test area with absorbent paper to remove excess sample solution, and then use a skin moisture meter to measure the skin moisture content of the subject.

[0046] ⑤ Record the experimental results and perform data analysis. As shown in Table 2.

[0047] Table 2: Results of Moisturizing Performance Evaluation of Various Recombinant Type III Humanized Collagen and Sodium Alginate Complexes

[0048] As shown in Table 2, the moisturizing improvement rate of each complex is: Complex 1 of this invention > Complex 2 of this invention > Complex 3 of this invention > Comparative complex. This indicates that the complexes after different enzymatic digestion and cross-linking all improve the skin's moisturizing rate. Among them, the complex solution prepared in Example 1 with the largest molecular weight has the most significant moisturizing improvement rate, and the P value is <0.0001, which is extremely statistically significant.

[0049] 4) Cell adhesion performance test, the method is as follows: ① Use DMEM / F12 medium containing 10% serum and other components including penicillin (100 U / mL), streptomycin (100 μg / mL), and glutamine (2 mmol / L). Human dermal fibroblasts (HSF) were seeded into the medium at a density of 5 × 10^4 cells / mL and cultured in an incubator at 37°C and 5% CO2.

[0050] ② Preparation of sample solutions. Dilute the sample solutions of complexes 1-3 and the control complex of the present invention with cell culture medium to prepare a 10% (v / v) solution.

[0051] ③ Sample addition and culture. Add 100 μL of sample solution to a 96-well plate, with three replicates for each concentration, and incubate in a cell culture incubator for 4 hours. Collect HSF cells in the logarithmic growth phase, adjust the cell suspension concentration, add 100 μL to each well of the 96-well plate, with 2000-3000 cells per well, and incubate in a cell culture incubator for 2 hours. Simultaneously, set up a blank control group without sample solution.

[0052] ④ Discard the cell culture medium in the well plate, add 200 μL of cell culture medium containing 10% fluorescent staining agent, cover with aluminum foil, and incubate at 37℃ in a 5% CO2 incubator for 1 h. Cover the well plate with sealing film, observe and record the initial cell count (Nc) using an inverted fluorescence microscope, centrifuge at 1000 rpm for 5 min, discard the sealing film and supernatant, wash once with PBS buffer, add 100 μL of washing solution, and count the adsorbed cells (Nt). Adhesion rate X = Nt / Nc × 100%. The results are shown in Table 3 and... Figure 4 As shown.

[0053] Table 3: Results of cell adhesion performance evaluation of various recombinant type III humanized collagen complexes with sodium alginate

[0054] From Table 3 and Figure 4The results show that at concentrations of 10%, 5%, and 1%, the cell adhesion rates of complexes 1-3 of the present invention are all higher than those of the control complex, and the cell adhesion rate of complex 1 of the present invention is higher than that of complexes 2 and 3 of the present invention.

[0055] 5) Cell migration performance test, the method is as follows: ① Use DMEM / F12 medium containing 10% serum and other components including penicillin (100 U / mL), streptomycin (100 μg / mL), and glutamine (2 mmol / L). Human dermal fibroblasts (HSF) were seeded into the medium at a density of 5 × 10^4 cells / mL and cultured in an incubator at 37°C and 5% CO2.

[0056] ② Collect HSF cells in the logarithmic growth phase, centrifuge at 1000 rpm for 5 min, resuspend in complete culture medium and adjust the cell suspension concentration. Add 1.5 mL of cell suspension to each well of a 12-well cell culture plate, with 8 × 10⁵ cells per well, ensuring that the cells can completely cover the bottom of the well overnight. Each group has 3 replicates. Incubate at 37°C with 5% CO₂ for 24 h until a monolayer of cells completely covers the bottom of the well.

[0057] ③ Using a 200 μL pipette tip, quickly make incisions with a ruler. Wash with PBS to remove the incised cells. Replace the complete culture medium in the wells with 1.5 mL of serum-free culture medium containing 10% (v / v) of the present invention's complexes 1-3 and the control complex solution. A blank control group was also established. Each well was photographed using an inverted microscope to obtain the cell image at 0 h.

[0058] ④ Incubate the cells in a 5% CO2, 37°C incubator for 24 hours, then end the experiment and take photos. Process the photos using software to calculate the cell migration rate in the scratched area of ​​each photo.

[0059] ⑤ Migration rate calculation. Migration rate (%) = S1 / S0 × 100%; Where: S1 is the area occupied by cells in the scratch region at 24h, and S0 is the total area of ​​the scratch region at 0h.

[0060] The results are shown in Table 4 and Figure 5 As shown.

[0061] Table 4: Results of Cell Migration Performance Evaluation of Various Recombinant Type III Humanized Collagen and Sodium Alginate Complexes

[0062] From Table 4 and Figure 5The results also show that the cell migration performance of the composites 1-3 prepared in Examples 1-3 is higher than that of the control composites, and the cell migration performance of composite 1 prepared in Example 1 is higher than that of composites 2 and 3 prepared in Examples 2 and 3.

[0063] Example 4 This embodiment investigates the effect of the mass ratio of recombinant type III humanized collagen microspheres to sodium alginate on product performance.

[0064] Recombinant type III humanized collagen microspheres and sodium alginate were dissolved in 0.01 M phosphate buffer at concentrations of 1.0 g / L and 10 g / L (mass ratio 1:10), 0.5 g / L and 10 g / L (mass ratio 1:20), 0.25 g / L and 10 g / L (mass ratio 1:40), and 0.1 g / L and 10 g / L (mass ratio 1:100), respectively. The preparation process of the complex was the same as in Example 1.

[0065] Viscosity test method: Take 200 ml of the complex solution and place it in a 250 ml beaker. Use a rotational viscometer with rotor No. 1 and a speed of 3 rpm to measure the viscosity. The results are shown in Table 5.

[0066] Table 5: Effect of the mass ratio of recombinant type III humanized collagen to modified sodium alginate on product viscosity

[0067] As shown in Table 5, with the sodium alginate concentration remaining constant, the viscosity test results at mass ratios of 1:10 and 1:20 were not significantly different, indicating that increasing the amount of recombinant type III humanized collagen microspheres did not significantly increase the viscosity of the complex. The viscosity test results at mass ratios of 1:40 and 1:100 were 941 mPa·s and 867 mPa·s, respectively, both significantly lower than the viscosity test results at 1:20. Therefore, a mass ratio of 1:20 for recombinant type III humanized collagen to modified sodium alginate, i.e., an addition amount of 0.5 g / L and 10 g / L, was selected for preparing the complex solution.

[0068] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and not restrictive in all respects. Furthermore, it should be understood that although this specification describes embodiments, it does not encompass only one technical solution. This descriptive method is merely for clarity, and those skilled in the art should consider the specification as a whole. The technical solutions in the embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A recombinant type III humanized collagenase-cleaved peptide-oxidized sodium alginate complex, characterized in that: The complex is formed by dissolving recombinant type III humanized collagen microspheres and oxidized modified sodium alginate in phosphate buffer at a mass ratio, adding specific protease to enzymatically cleave the recombinant type III humanized collagen microspheres to form specific polypeptide fragments, adjusting the pH of the reaction solution to 9-10 and the temperature to 55-65℃, so that the polypeptide fragments obtained by enzymatic cleavage are directly cross-linked with the oxidized modified sodium alginate. The specific protease is selected from: Glu-C alone, or a mixture of Lys-C and Arg-C; The method for preparing the oxidized modified sodium alginate is as follows: sodium alginate is dispersed in water, sodium periodate is added, and the mixture is reacted at 3-5°C in the dark for 15-18 hours. Then, ethylene glycol is added and the mixture is reacted at room temperature for 50-70 minutes to obtain a precipitate. The precipitate is then washed successively with water and anhydrous ethanol, and dried under vacuum at room temperature for 6 hours. The mass ratio of sodium alginate, sodium periodate, and ethylene glycol is 1:0.1:

5. The mass ratio of the recombinant type III humanized collagen microspheres to oxidized modified sodium alginate is 1:(10-20). The molecular weight of the oxidized sodium alginate is 150kDa to 200kDa, and the mass concentration of the oxidized sodium alginate in the composite is 10g / L. The system enzyme activity of the specific protease is 100 IU / L to 200 IU / L.

2. A method for preparing the recombinant type III humanized collagenase-cleaved peptide-oxidized sodium alginate complex according to claim 1, characterized in that: Includes the following steps: Step 1: Dissolve recombinant type III humanized collagen microspheres and oxidized modified sodium alginate in phosphate buffer solution according to the following mass ratios; Step 2: Add a specific protease to carry out the enzymatic digestion reaction; Step 3: Adjust the pH of the reaction solution to 9-10 and the temperature to 55-65℃ to carry out the cross-linking reaction and obtain the enzymatically digested protein peptide-sodium alginate complex solution; Step 4: The enzymatically digested protein peptide-sodium alginate complex solution is subjected to ultrafiltration for impurity removal and concentration, while simultaneously undergoing solution replacement until the pH value of the concentrate reaches the specified range, thereby obtaining the enzymatically digested protein peptide-sodium alginate complex concentrate. The ultrafiltration for impurity removal and concentration is performed using a 10kDa ultrafiltration membrane for retention, and the retentate is collected. The solution replacement is performed by adding a 0.01M phosphate buffer solution with a pH of 6.8 during ultrafiltration to replace the retentate solution system, adjusting the pH value of the retained concentrate to 7.0-8.

0. Step 5: Add water to the concentrated solution of the enzymatically digested protein peptide-sodium alginate complex to a certain volume.

3. The method for preparing the recombinant type III humanized collagenase-oxidized sodium alginate complex according to claim 2, characterized in that: In step one, the concentration of the phosphate buffer solution is 0.01M.

4. The method for preparing the recombinant type III humanized collagenase-oxidized sodium alginate complex according to claim 2, characterized in that: In step two, the enzyme digestion reaction time is controlled within 4 hours, the enzyme digestion reaction temperature is 37°C, and the reaction pH value is the optimal pH for the specific protease.

5. The method for preparing the recombinant type III humanized collagenase-oxidized sodium alginate complex according to claim 2, characterized in that: In step three, the solution used to adjust the pH of the reaction solution is a 6M NaOH solution; the cross-linking reaction takes 1.5 to 3 hours.

6. The application of the recombinant type III humanized collagenase-oxidized sodium alginate complex according to claim 1, characterized in that: Applications in the preparation of products for adjuvant treatment of skin and mucous membrane damage repair, or applications in the preparation of active ingredients for adjuvant treatment products for skin and mucous membrane damage repair.

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